Methods for identifying an essential gene in a prokaryotic microorganism

ABSTRACT

Methods are provided for the rapid identification of essential or conditionally essential DNA segments in any species of haploid cell (one copy chromosome per cell) that is capable of being transformed by artificial means and is capable of undergoing DNA recombination. The BAC system is used to provide to the prokaryotic host cell an additional copy of a known segment of DNA of the host cell (or of another prokaryotic cell whose genome is known) to construct merodiploid test cells wherein the chromosomal region of the host cell that is homologous to the DNA contained in the BAC becomes diploid. Alternatively, due to the high homology in essential genes of prokaryotes, the DNA contained in the BAC can be derived from a prokaryote other than the host cell. A transposon is then delivered randomly to the merodiploid cell. Due to presence of the BAC carrying a segment of homologous DNA, the merodiploid cell will survive replication if the transposon disrupted gene on the host chromosome is replaced by a second normal gene existing on the particular BAC contained within the particular host cell. However, in the event the BAC carrying the second normal gene copy is lost during replication or the BAC replaces a normal gene in the host cell with a defective copy due to recombination, inhibition of growth or lethality of the test cell will result. This system offers an enhanced means of identifying essential function genes in diploid pathogens, such as gram-negative and gram-positive bacteria.

[0001] This application claims priority under 35 U.S.C. § 119(e)(1) toU.S. provisional application Ser. No. 60/214,621, filed Jun. 28, 2000,which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field Of The Invention

[0003] The present invention relates generally to genetically engineeredcells, and more specifically, to the study of essential genes inprokaryotic microorganisms.

[0004] 2. Background Information

[0005] The discovery and development of drugs able to prevent and curebacterial infections have represented one of the Twentieth Century'smajor contributions toward improving human longevity and quality oflife. Antibacterial agents are among the most commonly prescribed drugsof any kind worldwide. Used properly and appropriately, these drugs arelifesaving. However, their indiscriminate use drives of the cost ofhealth care and leads to bacterial resistance that renders valuabledrugs useless. The rational use of antibacterial agents is dependent,among others, on an understanding of their mechanisms of action andbacterial strategies for resistance to drugs.

[0006] Antibacterial agents, like all anti-microbial drugs, are directedagainst unique targets not present in mammalian cells. The goal is tolimit toxicity to the host and maximize chemotherapeutic activityaffecting invading microbes only. One major difference between bacterialand mammalian cells i the presence in bacteria of a rigid wall externalto the cell membrane. The wall protects bacterial cells from osmoticrupture because of the difference between the hyperosmolar (up to 20atm) cell interior and the usually isosmolar or hyposmolar hostenvironment. In both gram-positive and gram-negative bacteria,peptidoglycan, a large, covalently linked sacculus that surrounds thebacterium, is the structure that confers cell wall rigidity andresistance to osmotic lysis. In gram positive bacteria, peptidoglycan isthe only later external to the cell membrane and is thick) 20 to 80 nm);while in gram-negative bacteria the peptidoglycan layer is thin (1 nm)and is protected by an outer membrane. Chemotherapeutic agents directedat any stage of the synthesis, export, assembly, or cross-linking ofpeptidoglycan inhibit bacterial cell growth and, in most cases, lead tocell death.

[0007] Bacitracin, a cyclic peptide antibiotic, inhibits the conversionto its active form of the lipid carrier that moves the water-solublecytoplasmic peptidoglycan subunits through the cell membrane to the cellexterior. Cell wall subunits accumulate in the cytoplasm and can beadded to the growing peptidoglycan chain.

[0008] Cyclopeptides, such as vancomycin and teichoplanin) are highmolecular weight antibiotics that bind to the terminalD-alanine-D-alanine component of the stem peptide when the subunits areexternal to the cell membrane and still linked to the lipid carrier.This binding sterically inhibits the addition of sub units to thepeptidoglycan backbone.

[0009] β-Lactam antibiotics, such as penicillins, cephalosporins,carbapenems, and monobactams, characterized by a four-membered β-Lactamring, prevent the cross- linking reaction called transpeptidation.Energy for attaching a peptide cross-bridge from the stem peptide of onepeptidoglycan subunit to another is derived from the cleavage of aterminal D-alanine residue from the subunit stem peptide. The β-Lactamring of the antibiotic forms an irreversible covalent acyl bond with thetranspeptidase enzyme, preventing the cross-linking reaction.Transpeptidases and similar enzymes involved in cross-linking are calledpenicillin-binding proteins because they have active sites that bindβ-Lactam antibiotics.

[0010] Virtually all the antibiotics that inhibit bacterial cell wallsynthesis are bactericidal, eventually resulting in the cell's death dueto osmotic lysis. However, much of the loss of cell wall integrityfollowing treatment with cell -active agents is due to the bacteria'sown cell wall-remodeling enzymes (autolysins) that cleave peptidoglycanbonds in the normal course of cell growth. Autolysis without normal cellwall repair results in weakness and eventual cell death.

[0011] Most of the antibacterial agents that inhibit protein synthesisinteract with the bacterial ribosome. The differences between thecomposition of bacterial and mammalian ribosomes give these compoundstheir selectivity. For example, aminoglycosides are a group ofstructurally related compounds containing three linked hexose sugars.Aminoglycosides exert a bactericidal effect by binding irreversibly tothe 30 S subunit of the bacterial ribosomes and blocking initiation ofprotein synthesis. Macrolides and lincosamides, although structurallydifferent, are two types of antibiotics that bind specifically to the 50S portion of the bacterial ribosome. Chloramphenicol also bindsirreversibly to the 50 S portion of the bacterial ribosome at a siteclose but not identical to the sites binding the macrolides andlincosamides. Tetracyclines interact reversibly with the bacterial 30 Sribosomal subunit, blocking the binding of aminoacyl tRNA to themRNA-ribosome complex. This mechanism is markedly different from that ofthe aminoglycosides, which also bind to the 30 S subunit.

[0012] The antimetabolites are all synthetic compounds that interferewith bacterial synthesis of folic acid. Products of the folic acidsynthesis pathway function as coenzymes for the one-carbon transferreactions that are essential for the synthesis of thyrnidine, allpurines, and several amino acids. Inhibition of folate synthesis leadsto cessation of cell growth and in some cases, to bacterial cell death.The principal antibacterial antimetabolites are sulfonamides andtrimethoprim.

[0013] Numerous additional antibacterial compounds have disparateeffects on nucleic acids. The quinolones, including nalidixic acid andits fluorinated derivatives, are synthetic compounds that inhibit theactivity of the A subunit of the bacterial enzyme DNA gyrase, which isresponsible for negative supercoiling of DNA during replication in theintact cell. The antibiotic novobiocin also interferes with the activityof DNA gyrase, but it interferes with the B subunit. Rifampin, usedprimarily as an antituberculosis agent, binds tightly to bacterialDNA-dependent RNA polymerase, thus inhibiting transcription of DNA intoRNA, and nitrofurantoin, a synthetic compound, causes DNA damage, beingreduced by a bacterial enzyme to highly reactive, short-livedintermediates that are thought to cause DNA strand breakage.

[0014] Still other compounds cause alternation of cell membranepermeability. The polymyxins behave as cationic, surface-activecompounds that disrupt the permeability of both the outer and thecytoplasmic membranes of gram-negative bacteria. Gramicidin A, on theother hand, acts as an ionophore, forming pores or channels in lipidbilayers.

[0015] One major and important class of genes consists of thosebacterial genes that are essential for growth or viability of abacterium. Because useful conventional antibiotics, such as thosedescribed above, are known to act by interfering with the products ofessential genes, it is likely that the discovery of new essential geneproducts will have a significant impact on efforts to develop novelantimicrobial drugs.

[0016] Conditional mutations such as temperature or suppressor sensitivemutations have been used in the past to identify some of the essentialgenes in bacteria. However, not all essential genes can be identified bythese types of mutations. The limitation is due to the fact that theseconditional mutations must occur at a specific codon of the genes inorder to alter the coded amino acid of the protein. Therefore, theoccurrence of mutants with these phenotypes is expected to be low.Moreover, not all of the gene can be converted to conditional mutations;there may be no codon causing these mutations in their nucleotidesequences.

[0017] Essential gene products have been traditionally identifiedthrough the isolation of conditional lethal mutants, or by transposonmutagenesis in the presence of a complementing wild type allele(balanced lethality). However, such approaches are laborious, as theyrequire identification, purification, and study of individual mutantstrains. These methods are also limited to species with well-developedsystems for genetic manipulation and, therefore, cannot be readilyapplied to many of the potentially dangerous microorganisms whosegenomes have recently been sequenced.

[0018] For example, conditional mutations such as temperature orsuppressor sensitive mutations have been used in the past to identifysome of the essential genes in bacteria. However, not all of theessential genes can be identified by these types of mutations becausethe conditional mutation must occur at a specific codon of the genes inorder to alter the coded amino acid of the protein. Therefore, theoccurrence of mutants with these phenotypes is expected to be low.Moreover, not all of the gene can be converted to conditional mutations;there may be no codon causing the conditional mutation in the nucleotidesequences of the bacterial genome.

[0019] Thus, it can be seen that successful antibiotics are allcompounds that in one way or another impair function of an essentialgene in bacteria. Despite the great discoveries that have been made,there is great need for new and better antibiotic compounds forpharmaceutical use in the treatment of mammalian species, including farmanimals, pets and humans and for development of laboratory methodsuseful in development of such new compounds. The present inventionsatisfies this need and provides additional advantages.

SUMMARY OF THE INVENTION

[0020] In one embodiment according to the present invention, there areprovided methods for identifying an essential chromosomal gene in ahaploid test organism utilizing a BAC-carrying merodiploid test cellconstructed from a wild-type haploid host cell that is transformed witha bacterial artificial chromosome (BAC) carrying a segment of DNA of thehaploid test organism. The segment of DNA in the BAC is homologous to aknown segment of chromosomal DNA in the host cell and the BAC isengineered to be sensitive to an environmental condition thatselectively prevents replication of the BAC in the host cell. Abacterial transposon is then inserted into the merodiploid test cell soas to disrupt function of a gene therein and one or more of theBAC-carrying merodiploid test cells are cultured in a suitable culturemedium while introducing the environmental condition so as to transformthe merodiploid test cells into haploid test cells. One or more of thehaploid test cells that contain transposon-mutagenized DNA in anessential chromosomal gene therein are identified.

[0021] In another embodiment according to the present invention, thereare provided methods for screening bacterial genes in a pathogenicbacterium whose genome is known to select compounds with putativeantibiotic activity. In this embodiment, a BAC-carrying merodiploid testcell is constructed by transforming a wild-type haploid host cell with aBAC that carries a known segment of DNA of a pathogenic bacterium, whichsegment is homologous to a segment of chromosomal DNA in the host cell,and wherein the BAC in the test cell is sensitive to an environmentalcondition that selectively prevents replication of the BAC in the testcell. A transposon transposon is randomly inserted into the merodiploidtest cell so as to disrupt function of a gene therein and one or more ofthe merodiploid test cells are cultured in a suitable culture mediumwhile introducing the environmental condition. One or more test cellsthat do not survive subjection to the environmental condition areidentified as containing the transposon in an essential chromosomal genetherein and a corresponding essential gene is obtained in the knownsegment of DNA of the pathogenic bacterium by homology with theidentified essential chromosomal gene in the test cell. Thecorresponding essential gene obtained from the pathogenic bacterium or abacterial protein encoded by the corresponding essential gene isscreened against putative antibiotic compounds to determine thosecompounds that bind to or interrupt function of the correspondingessential gene or the bacterial protein. Such a compound is a candidateantibiotic against the pathogenic bacterium.

[0022] In yet another embodiment according to the present invention,there are provided methods for identifying an essential chromosomal genein a haploid test organism. In this embodiment, the invention methodcomprises constructing a BAC carrying a known segment of DNA of thehaploid test organism, which segment is homologous to a known segment ofchromosomal DNA in a haploid host cell and randomly inserting abacterial transposon into the BAC so as to disrupt function of a gene inthe segment of chromosomal DNA. The BAC is then introduced into thehaploid test cell to create a merodiploid test cell, the merodiploidtest cell is cultured in a suitable culture medium, and one or moreBAC-carrying merodiploid test cells that do not survive in culture isidentified as containing the transposon in an essential chromosomal genetherein. Identity of the essential chromosomal gene is obtained byhomology with the known segment of DNA inserted into the BAC that wasintroduced into the identified test cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 is a schematic diagram of the invention method wherein anE. coli host cell is transfected with a temperature sensitive BACcontaining a segment of DNA of a test cell to create a merodiploid cell.A transposon is introduced into an essential gene of the E. coli withinthe region of host chromosomal DNA having homology with the segment ofDNA of the test cells. Incubation of the merodiploid cell at 30° C. doesnot affect viability, but incubation at 43° C. kills the temperaturesensitive BAC. Death of the merodiploid cell indicates that thetransposon disrupted an essential gene in the E. coli DNA causing deathof the host cell.

[0024]FIG. 2 is a schematic diagram showing preparation of a merodiploidtest cell using a BAC (medium-sized open circle) into which Tn5 (shownas a small dark circle) is randomly inserted to form BAC-Tn5. A hostcell (shown as a square with a large open circular chromosomal DNA) isthen transformed with BAC-Tn5.

[0025]FIG. 3 is a schematic diagram illustrating linearization ofBAC-Tn5 in preparation for insertion into a host cell byelectroporation.

[0026]FIG. 4 is a schematic diagram comparing two outcomes of insertionof the linearized BAC-Tn5t into E. coli. In the top representationBAC-Tn5 does not contain a mutation in an essential gene and the hostcell survives replacement of genomic DNA by homologous DNA in BAC-Tn5.In the bottom scheme BAC-Tn5 does contain a mutation in a gene thatcorresponds to an essential gene in host chromosomal DNA and the hostcell does not survive replacement of its genomic DNA by homologous DNAin BAC-Tn5.

DETAILED DESCRIPTION OF THE INVENTION

[0027] A new method has been developed, as described here, to identifyand determine genes that are essential for growth of prokaryotes.Escherichia coli, one of the most extensively studied prokaryotes, isused as a model microorganism. The essential genes are genes requiredfor synthesis of macromolecules such as proteins, RNA and DNA; forconstruction of cell wall, membrane, ribosomes and other structuralcomponents; and for other survival functions. Functional loss of any ofthese genes causes growth inhibition even in nutrient rich media, andeven eventual cell death. Genes for synthesis of amino acids, vitaminsand other organic/inorganic materials are generally not essential genes.Since these small compounds are readily found in nutrient rich media,defective genes for these nutrient syntheses do not lead to cell death.

[0028] The present invention is based on the knowledge that duringevolution the essential genes in most of the prokaryotic microorganismsare conserved as analogous genes (orthologs) in their genomes.Therefore, for example, the essential genes found in E. coli are verysimilar, i.e., homologous, to the corresponding essential gene in avariety of other bacterial species, including bacterial pathogens. As aresult, E. coli, or another of the prokaryotic microorganisms whosegenome has been completely mapped, can be used as a framework to developantibiotics that act with broad spectrum and high efficacy byinactivation of an essential gene or essential gene product common to avariety of pathogenic bacteria. Because the target proteins are coded bygenes indispensable to cell growth, antibiotics to be developed usingthe invention methods disclosed herein will generally be bactericidalrather than bacteriostatic. The proteins coded for by the essentialgenes of one prokaryote are ideal target molecules for identificationand development of antibiotics against diploid infectious bacterialpathogens.

[0029] Prokaryotic organisms, such as E. coli, are haploid cells (onecopy chromosome per cell) while they are not replicating. Consequently,when a potentially lethal mutation by transposon insertion occurs in oneof the essential genes, the resultant prokaryotic cell dies, making itimpossible to recover the mutated bacteria as a viable cell foridentification and analysis of the essential gene discovered byknockout. The difficulty posed by this phenomenon is overcome in theinvention method by providing to the test cell an additional copy of thegene that will be knocked out before the mutation occurs. Thus, thesecond gene can supply the normal function, and the cell can survivedespite the presence of the lethal mutation.

[0030] The invention methods, as described herein, employ two keytechnologies; a bacterial transposon, such as Tn10 or Tn5, for knockoutmutagenesis and the BAC (Bacterial Artificial Chromosome) cloning systemfor constructing partially diploid cells (merodiploids) or fortransferring a knocked out gene in a segment of DNA whose sequence isknown from the BAC to a host cell. A transposon is a DNA molecule thatis capable of integrating into a target DNA molecule, without sharinghomology with the target DNA molecule. The target molecule may be, forexample, chromosomal DNA, cloned DNA, or PCR-amplified DNA. Transposonintegration is catalyzed by transposase enzyme, which may be encoded bythe transposon itself, or may be exogenously supplied. One example of atransposon is mariner. Other examples include Tn5, Tn7 and Tn10. In thepresent invention, the transposon is used to inactivate normal functionof a gene by insertion and by subsequent disruption of the correctreading frame of the gene. The insertion occurs randomly on host cellchromosome or in the segment of test cell DNA contained in a BAC and anyinsertion in a gene makes the gene nonfunctional.

[0031] Accordingly, in one embodiment according to the presentinvention, there are provided methods for identifying an essential genein a haploid test organism. In the invention method a BAC-carryingmerodiploid test cell is constructed by transforming a wild-type haploidhost cell with a bacterial artificial chromosome (BAC) carrying a knownsegment of DNA of the haploid test organism carrying a segment of DNA ofthe haploid test organism, which segment is homologous to a knownsegment of chromosomal DNA in the host cell, and wherein replication ofthe BAC in the test cell is sensitive to an environmental condition thatselectively prevents replication of the BAC in the host cell, insertingrandomly a bacterial transposon into the merodiploid test cell so as todisrupt function of a gene therein, and culturing one or more of theBAC-carrying merodiploid test cells in a suitable culture medium whileintroducing the environmental condition so as to transform themerodiploid test cells into haploid test cells. The invention methodfurther comprises identifying one or more of the haploid test cellscontaining transposon-mutagenized DNA of an essential chromosomal geneand obtaining the essential chromosomal gene by homology with a gene inthe known segment of DNA. For example, test cells that contain atrasposon-mutagenized essential chromosomal gene do not grow in orsurvive subjection to growth conditions that are non-permissive togrowth of the BAC contained therein.

[0032] It is an important feature of the invention methods that themerodiploid host cell(s) are engineered to contain a BAC with a segmentof DNA that corresponds to (i.e., is homologous to) a known segment ofchromosomal DNA of the host cell. Homology between correspondingsegments of host cell and test organism DNA is typically in the rangefrom about 80% sequence identity to about 100% sequence identity, forexample, 85% identity, 90% identity or 95% identity. Sequencecorrespondence (i.e., homology as described above) between the segmentof DNA placed into an individual BAC and a particular segment ofchromosomal DNA of the host cell is established prior to construction ofthe merodiploid cells. For convenience, therefore, the host cell ispreferably a prokaryotic cell whose entire genome has been mapped sothat the location of each gene therein is known.

[0033] Due to the high degree of sequence conservation in essentialgenes among haploid organisms, the same organism can be used both ashost cell and as provider of the various segments of DNA that areinserted into the BACs. Alternatively, two different species of haploidorganism can be used as host cell and as provider of the varioussegments of DNA that are inserted into the BACs used in the inventionmethods. Preferred host cells for use in constructing the merodiploidcells used in the invention methods are E. coli, Salmonella, and B.subtilis, with E. coli being most preferred due to the extensiveknowledge of its genome.

[0034] In one embodiment according to the present invention, the BACsare introduced into the host cells in vivo using known methods fortransforming live cells with a plasmid. For example, the BAC may befirst introduced into a cosmid/fosmid and then the cosmid/fosmidintroduced directly into the host cell. Alternatively, thecosmid/fosmids may be packaged into a phage for efficiency of cloning inthe host cells. A preferred phage for this purpose is lambda phage. Whenthe BACs are introduced into the host cells in vivo to create amerodiploid cell, the transposon is randomly introduced into thecompleted merodiploid cell and may insert itself into any location inthe BAC or in the host cell.

[0035] To make removal of the BAC from the merodiploid test cellconditional, it is preferred to utilize a mutant BAC that is engineeredto be replication sensitive in maintenance. Such a replication sensitivemutant is readily eliminated from the cell by providing the condition toBAC is replication sensitive, e.g. a non-permissive temperature or areplication suppressor. When this happens, if the transposon disrupts anessential gene in host cell chromosomal DNA, the cell dies due to theloss of the undisrupted essential gene contained within its replicationsensitive BAC.

[0036] Therefore, in this embodiment of the invention, the BACs areengineered to be sensitive to an environmental condition, such thatculture of the BAC-containing host cell under the environmentalcondition prevents or inhibits replication of the BAC without killingthe host cell. In a preferred embodiment, the BAC may contain an originof replication having a mutation which makes its functioningtemperature-sensitive in the host cell. See, for example, Ehrlich, Proc.Natl. Acad. Sci. USA (1978) 75:1433. As used herein, a BAC containingsuch a mutation is referred to as being “temperature sensitive toreplication.” During replication of the BAC-containing host cell at thepermissive low temperature, for example 30° C. to 34° C., the BACremains viable in the host cell, but when the temperature is elevated toa non-permissive temperature, for example 45° C., the BAC ceases toreplicate and is lost from the host cell. See also U.S. Pat. No.5,925,544, which is incorporated herein by reference in its entirety.

[0037] To aid in determining whether the transposon inserts into anessential chromosomal gene of the host cell, the transposon ispreferably operably linked with a DNA sequence encoding a firstphenotypically selectable marker prior to its random insertion into themerodiploid cell. By “selectable marker” is meant a gene that alters theability of a cell harboring the transposon to grow or survive in a givengrowth environment relative to a similar cell lacking the selectablemarker. Such a marker may be a positive or negative selectable marker.For example, a positive selectable marker (e.g., an antibioticresistance or auxotrophic growth gene) encodes a product that confersgrowth or survival abilities in selective medium (e.g., containing anantibiotic or lacking an essential nutrient). A negative selectablemarker, in contrast, prevents transposon- harboring cells from growingin negative selection medium, when compared to cells not harboring thetransposon. A selectable marker may confer both positive and negativeselectability, depending upon the medium used to grow the cell. The useof selectable markers in prokaryotic cells is well known by those ofskill in the art.

[0038] Examples of such selectable markers that can be used for thispurpose in prokaryotic cells include antibiotic resistance genes forampicillin (β-lactamases), tetracycline, kanamycin, and chloramphenicol(chloramphenicol acetyltransferase). DNA encoding a secondphenotypically selectable marker can be included in the BAC such thatthe first and second markers are selected to be sensitive to twodifferent environmental conditions. Preferably, a second, different,antibiotic resistance gene is included in the BAC to allow fordetermination of whether the host cell has been successfully transformedwith a replication sensitive BAC. In practice of the invention methodwherein two different antibiotic resistance genes are used todistinguish clones that contain a transposon and a BAC, the identifyingincludes sequentially subjecting the test cells during culture to thefirst antibiotic and then to the second antibiotic to which theantibiotic resistance genes provide resistance.

[0039] In this embodiment of the invention, determination of tests cellswherein an essential gene in host chromosomal DNA has been “knocked out”depends upon the BAC being engineered to be replication sensitive tocause loss of the BAC contained within the merodiploid test cells bysubjecting the test cells to an environmental condition to which the BAChas been engineered to be replication sensitive. By this means, it isreadily determined whether loss of the BAC results in a lethalphenotype. When the test cells are grown at an elevated non-permissivetemperature under conditions that allow rapid detection of test cellviability, viability of a test cell (despite loss of the replicationsensitive BAC by its failure to replicate under non-permissiveconditions) is strong evidence that the randomly inserted transposon didnot become inserted into an essential gene in test cell chromosomal DNA.Conversely, test cells that are killed under these conditions areidentified as having had an essential gene in chromosomal DNA disruptedby the transposon. Due to the known homology between the segment of DNAcontained in the BAC and the corresponding segment of host chromosomalDNA, the exact location of the essential gene in the prokaryotic genomeis readily obtained, for example using various mapping methods known inthe art.

[0040] In another embodiment according to the invention, there areprovided methods for identifying an essential gene in a haploidorganism, for example a pathogenic bacterium, wherein the transposon israndomly introduced into the BAC before the BAC is introduced into thehost cell. In this embodiment of the invention methods, the transposonis randomly inserted into the BAC in vitro (either at the time the knownsegment of DNA is inserted into the BAC to form a transposon-containingBAC (BAC-Tn) or thereafter). By this process, which is sometimesreferred to in the art as “in vitro transposition,” the transposonintegrates into the known segment of DNA (e.g., the target DNA) in anon-living cell. In an in vitro transposition reaction, the transposonintegrates into the target DNA randomly, or with near randomness; thatis, all DNA regions in the known segment of DNA have approximately equalchances of being sites for transposon integration.

[0041] In at least some of the BAC-Tns so constructed, a knockout geneis formed within the known segment of DNA inserted into the BAC-Tn andthe BAC-Tn is used as an “allelic replacement vector” to introducemutations into the genome of a host cell by specific replacement of agene in the chromosome of the host cell with a mutated (i.e., knockout)copy. The BAC-Tn integrates into the host cell's chromosome byhomologous recombination (single cross-over) in a region where there isidentical or near-identical nucleotide sequence between the twomolecules. Homologous recombination is mediated by complementarybase-pairing, and may result in either insertion of the exogenous DNAinto the host target DNA (a single cross-over event), or replacement ofthe host target DNA by the exogenous DNA (a double cross-over event).

[0042] Preferably, the BAC-Tn contains a negative selectable markeroutside of the region of homology, appropriate selection yields cellsthat have lost the negative selection marker by a second homologousrecombination event (double cross-over) and contain only a mutant copyof the essential gene. Thus, recombination during replication is used toreplace a gene in the host cell chromosome with its homologous knockoutgene provided on the BAC-Tn. If the test cell does not survive inculture, it is identified as having had inserted into its chromosomalDNA a segment of DNA from its respective BAC-Tn wherein an essentialgene was disrupted by the transposon. Using this information and theknowledge of the particular segment of test organism DNA that wasintroduced into this particular test cell, the identity of the essentialgene is readily obtained using known methods. Preferably, the BAC-Tn isengineered to be antibiotic resistant so that selection of antibioticresistant clones will indicate test cells wherein the transposon did notinsert into an essential gene (although upon recombination antibioticresistance was conferred upon the test cell).

[0043] Unlike prior art methods for detection of essential genes inprokaryotes, in the invention method a complete gene is knocked out andmeans is provided for identifying the location in the genome of the testprokaryotic organism of the knocked- out gene. In a preferredembodiment, the invention method is used to identify virtually all ofthe essential genes in E. coli.

[0044] In host cells such as E. coli, a BAC containing up to 350 kb ofprokaryotic DNA can exist as a large plasmid. Thus, more than 100 genesof E. coli may be investigated in a single BAC clone. Since there areabout 4,289 open reading frames in E. coli chromosome, construction ofonly 50 to 100 merodiploid test cells containing different segments ofthe host cell DNA may be sufficient to complete the identification ofthe essential genes by the invention method.

[0045] In a preferred embodiment according to the present invention, theentire genome of the test organism is used in construction of the BACssuch that a library of merodiploid host cells collectively contains theentire genome of the test organism is constructed for testing accordingto the invention method. For this purpose, it is preferred to shear thegenome of the test organism to prepare blunt-ended segments having avery random distribution. The insertions can be verified and localizedby sequencing, PCR, Southern blot, cloning without restriction digestsusing terminal transferase, and the like.

[0046] For building a library of knock-out mutants, non -limitingexamples of two E. coli strains that can be used experimentally asdescribed herein, and that can be compared with respect to experimentalresults include: E. coli MG1655 (rph-1)(r_(k)+m_(k)+) and JM101 (supEthi-1 r_(k)+m_(k)+δ(lac-proAB)[F′ traD36 proAB lacI^(q)ZδM15].

[0047] To analyze the nature of insertion distribution throughout thegenome one can map the mutations on an E. coli matrix. The overalldistribution may be random and the transposed loci may be scattered(tranposition process is relatively random) with defined clustering ofthe related mutations (selection is biased) indicating a high density ofsequential insertions.

[0048] If desired, a complete knockout library can be built, archivedand made available in different formats. Preferably, to allow forstatistical analysis of the results obtained by the invention methods,sufficient of the merodiploid test cells are constructed to provide atleast quadruple coverage of the entire genome of the haploid testorganism. Using the invention methods, one can sequence and archiveenough mutants to reach complete saturation of a genome (or part of agenome).

[0049] Many of the gram-positive bacterial pathogens are diploidorganisms. Thus, the invention methods can be used for screeningbacterial genes in a pathogenic bacterium whose genome is known toselect compounds with putative antibiotic activity. In this embodiment,the invention screening methods comprise constructing BAC- carryingmerodiploid test cells as described above wherein a known segment of DNAof a pathogenic diploid bacterium is contained within the BACs. Theinvention screening methods further comprise obtaining the identity of acorresponding essential gene in the pathogenic bacterium by homologywith the identified essential chromosomal gene in the host cell, andscreening the corresponding essential gene obtained from the pathogenicbacterium or a bacterial protein encoded by the essential gene againstputative antibiotic compounds to determine those compounds that bind toor interrupt function of the essential gene or the bacterial proteinencoded thereby. Such a compound is a candidate antibiotic against thepathogenic bacterium and may include small molecules, a protein orpolypeptides or a polynucleotide.

[0050] Illustrative pathogenic positive bacterium that can be screenedto identify essential genes therein using the invention methods includesuch organisms as Actinobacillus actinomycetemcomitans; Borreliaburgdorferi; Chlamydia trachomatis; Enterococcusfaecalis; Escherichiacoli; Haemophilus influenzae; Helicobacter pylori; Legionellapneumophila; Mycobacterium avium; Mycobacterium tuberculosis; Mycoplasmagenitalium; Mycoplasma pneumonia; Neisseria gonorrhoeae; Neisseriameningitidis; Staphylococcus aureus; Streptococcus pneumoniae;Streptococcus pyogenes; Treponema pallidum; Vibrio cholerae, and thelike.

[0051] By “homology” is meant sequence similarity between two peptidesor between two nucleic acid molecules. Homology can be determined bycomparing the residues in positions in two sequences, which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid as the sequence towhich it is compared, then the molecules are homologous at thatposition. Percent homology between sequences is a function of the numberof matching or homologous positions shared by the sequences.

[0052] The sequence data of a test clone is aligned to the sequences inthe database or databases using algorithms designed to measure homologybetween two or more sequences. Sequence alignment methods include, forexample, BLAST (Altschul et al., 1990), BLITZ (MPsrch) (Sturrock &Collins, 1993), and FASTA (Person & Lipman, 1988). For example, optimalalignment of sequences for aligning a comparison window may be conductedby the local homology algorithm of Smith (Smith and Waterman, Adv ApplMath, 1981; Smith and Waterman, J Theor Biol, 1981; Smith and Waterman,J Mol Biol, 1981; Smith et al, J Mol Evol, 1981), by the homologyalignment algorithm of Needleman (Needleman and Wuncsch, 1970), by thesearch of similarity method of Pearson (Pearson and Lipman, 1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package Release 7.0,Genetics Computer Group, 575 Science Dr., Madison, WI, or the SequenceAnalysis Software Package of the Genetics Computer Group, University ofWisconsin, Madison, Wis.), or by inspection, and the best alignment(i.e., resulting in the highest percentage of homology over thecomparison window) generated by the various methods is selected.

[0053] By “permissive growth conditions” or “rich growth conditions” ismeant an environment that is relatively favorable for cell growth and/orviability. Such conditions take into account the relative availabilityof nutrients, the absence of toxins, and optimal temperature,atmospheric pressure, presence or absence of gases (such as oxygen andcarbon dioxide), and exposure to light, as required by the organismbeing studied. Permissive growth conditions may exist in vitro (such asin liquid and on solid culture media) or in vivo (such as in the naturalhost or environment of the cell being studied).

[0054] By “non-permissive growth conditions” is meant an environmentthat is relatively unfavorable for growth and/or viability of cells ofan organism. An unfavorable environment may be due to nutrientlimitations (e.g., as seen with “minimal” bacterial growth medium suchas MIc), the presence of a compound that is toxic for the cell understudy, an environmental temperature, gas concentration, light intensity,or atmospheric pressure that is extreme (e.g., either too high or toolow) for optimal growth/viability of the organism under study.

[0055] By “gene that is essential for growth and/or viability” or by“essential gene” or by “essential gene in a known segment of DNA” ismeant a DNA element such as an origin of replication or a gene thatencodes a polypeptide or RNA whose function is required for survival,growth, or mitosis/meiosis of a cell. Insertion of a transposon into anessential gene may be lethal, i.e., prevent a cell from surviving, or itmay prevent a cell from growing or undergoing mitosis/meiosis.Alternatively, insertion of a transposon into an essential gene mayallow survival of a cell, but result in severely diminished growth ormetabolic rate. An essential gene also may be conditionally essential(i.e., required for viability and/or growth under certain conditions,but not under other conditions).

[0056] By “absence of transposons” is meant that fewer transposoninsertions are detected in an essential region of DNA, relative to thenumber of transposon insertions detected in a non-essential region ofDNA. An absence of transposons may be absolute (i.e., zero transposonsdetected) or relative (i.e., fewer transposons detected).

[0057] By “transformation” or “transforming” is meant any method knownin the art for introducing foreign molecules, such as DNA, into a cell.Lipofection, DEAE-dextran-mediated transfection, microinjection,protoplast fusion, calcium phosphate precipitation, retroviral delivery,electroporation, natural transformation, and biolistic transformationare just a few of the methods known to those skilled in the art whichmay be used. For example, biolistic transformation is a method forintroducing foreign molecules into a cell using velocity drivenmicroprojectiles such as tungsten or gold particles. Suchvelocity-driven methods originate from pressure bursts that include, butare not limited to, helium-driven, air-driven, and gunpowder-driventechniques.

[0058] By “identifying cells containing transposon-mutagenized DNA of anessential gene” is meant exposing the population of cells transformedwith transposon- mutagenized DNA to selective pressure (such as growthin the presence of an antibiotic or the absence of a nutrient)consistent with a selectable marker carried by the transposon (e.g., anantibiotic resistance gene or auxotrophic growth gene known to thoseskilled in the art). Identifying cells containing mutagenized DNA mayalso be done by subjecting transformed cells to a reporter gene assayfor a reporter gene product encoded by the transposon. Selections andscreens may be employed to identify cells containing mutagenized DNA,although selections are preferred.

[0059] It will be understood by those of skill in the art that the“detectable genes” disclosed herein can be replaced by any gene whichencodes a product whose expression is detectable and/or quantitatable byimmunological, chemical, biochemical, biological, or mechanical assays.A reporter gene product may, for example, have one of the followingattributes, without restriction: fluorescence (e.g., green fluorescentprotein), enzymatic activity (e.g., lacZ/β-galactosidase, luciferase,chloramphenicol acetyltransferase, alkaline phosphatase), toxicity(e.g., ricin), or an ability to be specifically bound by a secondmolecule (e.g., biotin or a detectably labelled antibody). It isunderstood that any engineered variants of reporter genes, which arereadily available to one skilled in the art, are also included, withoutrestriction, in the foregoing definition.

[0060] By “obtaining an essential gene in a segment of DNA” is meantdetermining that a given stretch of DNA contains a gene that isnecessary for cell growth and/or viability and locating the essentialgene within the segment of DNA. Such a gene may be necessary under all,or only under some (e.g., stringent) growth conditions. The locating maybe done, for example, by such techniques as PCR footprinting, byutilizing primers to obtain copies by PCR and compare the copies ofgenes in the segment of DNA with wild type genes in the host cell, andthe like.

[0061] The invention provides a method for the rapid identification ofessential or conditionally essential DNA segments. The method isapplicable to any species of haploid cell (one copy chromosome per cell)that is capable of being transformed by artificial means and is capableof undergoing DNA recombination. This system offers an enhanced means ofidentifying essential function genes in pathogens, such as gram negativeand gram-positive bacteria.

[0062] The invention will be further described with reference to thefollowing examples; however, it is to be understood that the inventionis not limited to such examples.

EXAMPLE 1

[0063] Merodiploid cells test cells were prepared using E. coli as hostcell and as provider of DNA segments that were inserted into bacterialartificial chromosomes that were engineered to be temperature sensitivefor replication (BACts). The E. coli host cells were transformed withthe BACts and then the merodiploid cells were randomly transformed invivo with Tn5 transposon. Following insertion of Tn5 into themerodiploid cells, six different clones could be expected, as shown inTable 1 below, depending on the insertion location of Tn5 in themerodiploid test cells. The BACts and the transposon, Tn5, were taggedby Chloramphenicol (Cm) and Kanamycin (Km) resistance markers,respectively. The drug resistance markers were used to detect thepresence of BACts and Tn5 in the transformed cells. TABLE 1 Growth atGrowth at 30 C on LB + Growth at 43 C on Location of Tn5 KM + Cm 43 C onLB + LB plate 43 C + Clone Types Transposon plate Km plate (No drug) Cm

No Transposon No No Yes No

Transposon in non-essential gene outside BAC homology region Yes Yes YesNo

Transposon in essential gene outside BAC homology region No No No No

Transposon in essential gene outside BAC homology region Yes Yes Yes No

Transposon in essential gene outside BAC homology region Yes Yes Yes No

Transposon in BAC Yes No Yes No

[0064] Thus, the six different types of clones can be differentiated bycolony formation in the absence or presence of drugs in Luria broth (LB)media and by the temperature change from 30° C. to 43° C. For example,clone D can grow in the presence of Km and Cm at both 30° C. and 43° C.,indicating that the clone D has Tn5 inserted in a non-essential gene ofE. coli. On the other hand, clone E and F can grow in the presence ofboth Km and Cm only at 30° C., and the clones cannot grow at 43° C. inthe presence of Km. An ability to grow at 43° C. without the drugsdistinguish the two clones, indicating that Clone F has Tn5 in BACts andclone E has Tn5 in the essential gene in E. coli chromosome. Foridentification of the essential genes, type E clones were collected andstored.

[0065] BLAST searches were conducted to compare wild-type host DNA withchromosomal DNA of the type E clones to determine the location of themutagenized essential genes. These clones will be used forcharacterization of the essential genes. The proteins encoded by eachwild-type essential gene can then be produced in large amounts forantibiotic screening and development.

EXAMPLE 2

[0066] In an alternative method for preparation and screening ofmerodiploid BAC-carrying test cells, Tn5 was inserted into the BACts tocreate a temperature sensitive BAC designated BAC-Tn5. Generalrecombination during replication is used to replace a gene in the E.coli chromosome with its homologous knockout gene on BAC- Tn5. If theknockout gene in the BAC-Tn5 is not essential, the replacement becomessuccessful upon transformation, presenting viable Km resistant colonies.On the other hand, if the knockout occurred in an essential gene, whenreplacement is completed by recombination, it leads to no production ofKm resistant colony. In this way, one can identify essential genes byscoring Km resistant colonies.

[0067] In this method the BAC-Tn5 was constructed by insertion of Tn5into the BAC in vitro, instead of transposing Tn5 onto BAC in vivo asdescribed above in Example 1. The resultant BAC-Tn5 was used totransform E. coli. This process is illustrated in FIG. 3. In thisprotocol, each BAC-Tn5 already contains a Tn5 inserted therein in randomfashion before transformation of the host cells (i.e. E. coli). Inaddition, and unlike the method described in Example 1 above, there isonly one type of Km resistant clone for each Km resistant cell, a clonecontaining a knockout gene on BAC-Tn5 and its normal allele on the hostchromosome.

[0068] Although the method examines each BAC-Tn5 to identify essentialgenes on the BAC, whole genome-wide search is also possible. It involvesa construction of BACTn5 without having individual BAC clones prior tointroducing Tn5. This is accomplished by transposing Tn5 in vitro at thetime of ligating the various segments of test organism DNA thatrepresent the entire genome of the test organism into the BAC vectors(in this case known segments of E. coli DNA).

[0069] In order to increase the number of transformants formed by doublecrossover recombination, and to decrease the frequency of recombinantsformed by reciprocal Campbell type recombination, BAC-Tn5 DNA islinearized, the plasmid is removed, and E. coli host in which therecombination is carried out is a RecBC-SbcBC quadruple mutant (FIG. 4).

[0070] Although the invention has been described with reference to theabove examples, it will be understood that modifications and variationsare encompassed within the spirit and scope of the invention.Accordingly, the invention is limited only by the following claims.

What is claimed is:
 1. A method for identifying an essential chromosomalgene in a haploid test organism, said method comprising: constructing aBAC-carrying merodiploid test cell by transforming a wild-type haploidhost cell with a bacterial artificial chromosome (BAC) carrying asegment of DNA of the haploid test organism, which segment is homologousto a known segment of chromosomal DNA in the host cell, and whereinreplication of the BAC in the test cell is sensitive to an environmentalcondition that selectively prevents replication of the BAC in the hostcell; inserting randomly a bacterial transposon into the merodiploidtest cell so as to disrupt function of a gene therein; culturing one ormore of the BAC-carrying merodiploid test cells in a suitable culturemedium while introducing the environmental condition so as to transformthe merodiploid test cells into haploid test cells; and identifying oneor more of the haploid test cells that contain transposon- mutagenizedDNA in an essential chromosomal gene therein.
 2. The method of claim 1further comprising obtaining the essential chromosomal gene by homologywith a gene in the known segment of DNA.
 3. The method of claim 1wherein the identifying involves selection of test cells that do notsurvive subjection to the environmental condition as having thetransposon in an essential chromosomal gene therein.
 4. The method ofclaim 1, wherein the transposon is Tn5 or Tn10.
 5. The method of claim4, wherein the transposon is operatively linked to a first antibioticresistance gene.
 6. The method of claim 5, wherein the BAC comprises asecond antibiotic resistance gene, wherein the first and secondantibiotic resistance genes convey resistance to two differentantibiotic compounds.
 7. The method of claim 6, wherein the first andsecond antibiotic resistance genes are selected to provide resistance tothe group consisting of ampicillin, tetracycline, kanamycin, andchloramphenicol.
 8. The method of claim 7, wherein the first and secondantibiotic resistance gene provide resistance, respectively, tokanamycin and chloramphenicol.
 9. The method of claim 6, wherein theidentifying includes subjecting the test cells to both of theantibiotics to which the first and second antibiotic resistance genesprovide resistance.
 10. The method of claim 1, wherein the BAC istemperature sensitive for replication and the environmental condition isa temperature that is selectively non-permissive for replication of theBAC in the test cell.
 11. The method of claim 1, wherein the BAC issuppressor sensitive for replication and the environmental condition isa suppressor that selectively suppresses replication of the BAC in thetest cell.
 12. The method of claim 1, wherein the host cell is selectedfrom the group consisting of E. coli, Salmonellae, and B. subtilis. 13.The method of claim 12, wherein the host cell is E. coli.
 14. The methodof claim 1, wherein the identified essential chromosomal gene has 100%sequence identity with a gene in the known segment of DNA.
 15. Themethod of claim 1, wherein the identified essential chromosomal gene hasat least 90% sequence identity with a gene in the known segment of DNA.16. The method of claim 1, wherein the identified essential chromosomalgene has at least 80% sequence identity with a gene in the known segmentof DNA.
 17. The method of claim 1, wherein the BAC contains up to 100genes of the haploid test organism.
 18. The method of claim 1, whereinthe haploid test organism and the host cell are the same species ofprokaryote.
 19. The method of claim 1, wherein a library of theBAC-carrying merodiploid test cells is constructed such that the BACs inthe library collectively contain the entire genome of the haploid testorganism.
 20. The method of claim 19, wherein the entire genome of thehaploid organism is contained in about 50 to 100 merodiploid test cellsthat each contain a unique segment of the genome of the haploid testorganism.
 21. The method of claim 19, wherein the test cells in thelibrary are simultaneously subjected to the environmental condition. 22.The method of claim 1, wherein sufficient of the merodiploid test cellsare constructed to provide four-fold coverage of the entire genome ofthe haploid organism.
 23. The method of claim 1, wherein the BAC in themerodiploid test cell is contained within a fosmid/cosmid.
 24. Themethod of claim 23, wherein the fosmid/cosmid is packaged in lambdaphage prior to insertion into the host cell.
 25. A method for screeningbacterial genes in a pathogenic bacterium whose genome is known toselect compounds with putative antibiotic activity, said methodcomprising: constructing a BAC-carrying merodiploid test cell bytransforming a wild-type haploid host cell with a BAC that carries aknown segment of DNA of a pathogenic bacterium, which segment ishomologous to a segment of chromosomal DNA in the host cell, and whereinthe BAC in the test cell is sensitive to an environmental condition thatselectively prevents replication of the BAC in the test cell; insertingrandomly a transposon into the merodiploid test cell so as to disruptfunction of a gene therein; culturing one or more of the merodiploidtest cells in a suitable culture medium while introducing theenvironmental condition; identifying one or more test cells that do notsurvive subjection to the environmental condition as containing thetransposon in an essential chromosomal gene therein; obtaining acorresponding essential gene in the known segment of DNA of thepathogenic bacterium by homology with the identified essentialchromosomal gene in the test cell; and screening the correspondingessential gene obtained from the pathogenic bacterium or a bacterialprotein encoded by the corresponding essential gene against putativeantibiotic compounds to determine those compounds that bind to orinterrupt function of the corresponding essential gene or the bacterialprotein, wherein such a compound is a candidate antibiotic against thepathogenic bacterium.
 26. The method of claim 25, wherein the transposonis Tn5 or Tn10.
 27. The method of claim 25, wherein the transposon isoperatively linked to a first antibiotic resistance gene.
 28. The methodof claim 27, wherein the BAC comprises a second antibiotic resistancegene.
 29. The method of claim 28, wherein the first and secondantibiotic resistance genes are selected to provide resistance to thegroup consisting of ampicillin, tetracycline, kanamycin, andchloramphenicol.
 30. The method of claim 29, wherein the first andsecond antibiotic resistance gene provide resistance, respectively, tokanamycin and chloramphenicol.
 31. The method of claim 25, wherein theidentifying includes subjecting the test cells to the antibiotics towhich the first and second antibiotic resistance genes provideresistance.
 32. The method of claim 25, wherein the BAC is temperaturesensitive and the environmental condition is a non-permissivetemperature for replication of the BAC in the test cells.
 33. The methodof claim 25, wherein the BAC is suppressor sensitive and theenvironmental condition is a suppressor that selectively preventsreplication of the BAC in the test cells.
 34. The method of claim 25,wherein the host cell is selected from the group consisting of E. coli,Salmonellae, and B. subtilis.
 35. The method of claim 34, wherein thehost cell is E. coli.
 36. The method of claim 25, wherein the BACcontains up to 100 genes of the pathogenic bacterium.
 37. The method ofclaim 25, wherein a library of the BAC-carrying merodiploid test cellsis prepared such that the BACs in the library collectively contain theentire genome of the pathogenic bacterium.
 38. The method of claim 37,wherein the entire genome is contained in about 50 to 100 merodiploidtest cells that each contain a unique segment of the genome of thepathogenic bacterium.
 39. The method of claim 38, wherein the test cellsin the library are simultaneously subjected to the environmentalcondition.
 40. The method of claim 25, wherein the candidate antibioticis bactericidal.
 41. The method of claim 25, wherein the bacterium ispathogenic in at least one mammalian species.
 42. The method of claim25, wherein the bacterium is pathogenic in at least one plant species.43. A method for identifying an essential chromosomal gene in a haploidtest organism, said method comprising: constructing a BAC carrying aknown segment of DNA of the haploid test organism, which segment ishomologous to a known segment of chromosomal DNA in a haploid host cell;inserting randomly a bacterial transposon into the BAC so as to disruptfunction of a gene in the segment of chromosomal DNA; introducing theBAC into the haploid test cell to create a merodiploid test cell;culturing the merodiploid test cell in a suitable culture medium;identifying one or more BAC-carrying merodiploid test cells that do notsurvive in culture as containing the transposon in an essentialchromosomal gene therein; and obtaining the identity of the essentialchromosomal gene by homology with the known segment of DNA inserted intothe BAC that was introduced into the identified test cell.
 44. Themethod of claim 43, wherein the transposon is Tn5 or Tn10 .
 45. Themethod of claim 43, wherein the transposon is operatively linked to afirst antibiotic resistance gene.
 46. The method of claim 43, whereinthe transposon is inserted randomly into the BAC in vitro prior tointroduction of the BAC into the host cell.
 47. The method of claim 46,wherein the known segment of DNA is linearized prior to introductioninto the host cell.
 48. The method of claim 43, wherein the host cell isan E. coli RecBC-SbcBC quadruple mutant.
 49. The method of claim 43,wherein the identifying includes subjecting the test cells to theantibiotic to which the antibiotic resistance gene provides resistance.50. The method of claim 43, wherein the host cell is selected from thegroup consisting of E. coli, Salmonellae, and B. subtilis.
 51. Themethod of claim 43, wherein the BAC contains up to 100 genes of the testorganism.
 52. The method of claim 43, wherein a library of theBAC-carrying merodiploid test cells is prepared such that the BACs inthe library collectively contain the entire genome of the test organism.53. The method of claim 52, wherein the entire genome is contained inabout 50 to 100 merodiploid test cells that each contain a uniquesegment of the genome of the test organism.
 54. The method of claim 43,wherein the test organism is a pathogenic bacterium.
 55. The method ofclaim 54, wherein the method further comprises screening a correspondingessential gene obtained from the pathogenic bacterium or a bacterialprotein encoded thereby against putative antibiotic compounds todetermine those compounds that bind to or interrupt function of thecorresponding essential gene or the bacterial protein, wherein such acompound is a candidate antibiotic against the pathogenic bacterium.